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Restoration of mismatch repair to nuclear extracts of H6 colorectal tumor cells by a heterodimer of human MutL homologs. Proc Natl Acad Sci U S A

Duke University, Durham, North Carolina, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 04/1995; 92(6):1950-4. DOI: 10.1073/pnas.92.6.1950
Source: PubMed

ABSTRACT Hypermutable H6 colorectal tumor cells are defective in strand-specific mismatch repair and bear defects in both alleles of the hMLH1 gene. We have purified to near homogeneity an activity from HeLa cells that complements H6 nuclear extracts to restore repair proficiency on a set of heteroduplex DNAs representing the eight base-base mismatches as well as a number of slipped-strand, insertion/deletion mispairs. This activity behaves as a single species during fractionation and copurifies with proteins of 85 and 110 kDa. Microsequence analysis demonstrated both of these proteins to be homologs of bacterial MutL, with the former corresponding to the hMLH1 product and the latter to the product of hPMS2 or a closely related gene. The 1:1 molar stoichiometry of the two polypeptides and their hydrodynamic behavior indicate formation of a heterodimer, which we have designated hMutL alpha. These observations indicate that interactions between members of the family of human MutL homologs may be restricted.

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    • "This strongly supports the pathogenic potential of the p.Cys680Arg variant. The MLH1 protein exists predominately in a complex with PMS2 also known as the MutLa heterodimer (Li and Modrich 1995). The formation of a MutLa complex is essential for MMR activity (Baker et al. 1995, 1996; Edelmann et al. 1996) and, therefore, the failure of LS- MLH1 proteins to associate with PMS2 could result in "
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    ABSTRACT: In clinical genetic diagnostics, it is difficult to predict whether genetic mutations that do not greatly alter the primary sequence of the encoded protein causing unknown functional effects on cognate proteins lead to development of disease. Here, we report the clinical identification of c.2038 T>C missense mutation in exon 18 of the human MLH1 gene and biochemically characterization of the p.Cys680Arg mutant MLH1 protein to implicate it in the pathogenicity of the Lynch syndrome (LS). We show that the mutation is deficient in DNA mismatch repair and, therefore, contributing to LS in the carriers.
    07/2014; 2(4). DOI:10.1002/mgg3.80
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    • "In another approach, extracts from cell lines lacking a defined MMR protein are complemented with the missing MMR protein [Holmes et al., 1990; Li and Modrich, 1995]. The complemented extracts are added to an artificial substrate that carries a mismatch within a restriction site, interfering with its cleavage. "
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    ABSTRACT: Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes and is the most prevalent hereditary colorectal cancer syndrome. A significant proportion of variants identified in MMR and other common cancer susceptibility genes are missense or noncoding changes whose consequences for pathogenicity cannot be easily interpreted. Such variants are designated as "variants of uncertain significance" (VUS). Management of LS can be significantly improved by identifying individuals who carry a pathogenic variant and thus benefit from screening, preventive, and therapeutic measures. Also, identifying family members that do not carry the variant is important so they can be released from the intensive surveillance. Determining which genetic variants are pathogenic and which are neutral is a major challenge in clinical genetics. The profound mechanistic knowledge on the genetics and biochemistry of MMR enables the development and use of targeted assays to evaluate the pathogenicity of variants found in suspected patients with LS. We describe different approaches for the functional analysis of MMR gene VUS and propose development of a validated diagnostic framework. Furthermore, we call attention to common misconceptions about functional assays and endorse development of an integrated approach comprising validated assays for diagnosis of VUS in patients suspected of LS.
    Human Mutation 12/2012; 33(12). DOI:10.1002/humu.22168 · 5.05 Impact Factor
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    • "MutSc recognizes some base–base mismatches (Culligan and Hays, 2000; Wu et al., 2003) and it was also reported to play a role in meiotic recombination (Lloyd et al., 2007). Subsequent to DNA lesion recognition, MutS complexes, in the presence of ATP, interact with MutLa to direct DNA repair (Prolla et al., 1994; Li and Modrich, 1995). MutLa is a heterodimer of MLH1–PMS2 in humans and MLH1–PMS1 in yeast and plants. "
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    ABSTRACT: Deleterious effects of UV-B radiation on DNA include the formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). These lesions must be repaired to maintain the integrity of DNA and provide genetic stability. Of the several repair systems involved in the recognition and removal of UV-B-induced lesions in DNA, the focus in the present study was on the mismatch repair system (MMR). The contribution of MutSα (MSH2-MSH6) to UV-induced DNA lesion repair and cell cycle regulation was investigated. MSH2 and MSH6 genes in Arabidopsis and maize are up-regulated by UV-B, indicating that MMR may have a role in UV-B-induced DNA damage responses. Analysis of promoter sequences identified MSH6 as a target of the E2F transcription factors. Using electrophoretic mobility shift assays, MSH6 was experimentally validated as an E2F target gene, suggesting an interaction between MMR genes and the cell cycle control. Mutations in MSH2 or MSH6 caused an increased accumulation of CPDs relative to wild-type plants. In addition, msh2 mutant plants showed a different expression pattern of cell cycle marker genes after the UV-B treatment when compared with wild-type plants. Taken together, these data provide evidence that plant MutSα is involved in a UV-B-induced DNA damage response pathway.
    Journal of Experimental Botany 02/2011; 62(8):2925-37. DOI:10.1093/jxb/err001 · 5.79 Impact Factor
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